The continuing objective of the proposed studies is the elucidation of the biochemical mechanisms whereby chemicals produce liver cell necrosis. Pursuit of this objective employs 3 models: the liver cell death produced by galactosamine, phalloidin and bromobenzene. The first 2 agents are specific hepatotoxins that do not need to be metabolized by the mixed function oxidase system. Bromobenzene-induced liver cell injury has been believed to result from its metabolism by this system with the generation of an electrophilic epoxide that covalently binds to cellular macromolecules. Our studies of phalloidin and galactosamine suggest that both produce plasma membrane damage that results in the loss of the ability to maintain intra-cellular Ca2+ homeostasis. In both cases, we have shown that the plasma membrane injury is reversible in the absence of extracellular Ca2+ ions. Insight into the nature of this critical alteration in the plasma membranes has been gained, and the pursuit of the implication of these findings forms a major part of the specific objectives of the present application. A cell culture system utilizing hepatocytes from phenobarbital-induced rats has been developed that reproduces the essential features of bromobenzene-induced liver cell death. With this system, we have been able to dissociate the covalent binding of l4C-bromobenzene from the cell death, and an alternative mechanism for lethal cell injury was identified in the peroxidation of cellular phospholipids. We are proposing to study in greater depth the mechanisms of production of lipid peroxidation in bromobenzene-intoxicated hepatocytes as well as the role of this process in the liver cell death. Finally, lipid peroxidation has been observed with another important liver toxin, acetoaminophen. The implications of this observation are also to be pursued.